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Molecular mechanisms involved in the nucleolytic cleavage at the 18S rRNA/internal transcribed spacer 1 (ITS 1) junction, a late step of small-subunit pre-rRNA processing in vertebrates, remain largely unknown, mostly due to the lack of faithful in vitro assays. To identify the minimal cis-acting signals required for this reaction, we studied the processing of truncated human rRNA gene transcripts transiently expressed upon transfection of rRNA minigenes into cultured mouse cells. We observed that processing at this site was faithfully reproduced with transcripts containing only 60 nucleotides of 18S rRNA and the adjacent 103 nucleotides of ITS 1, but was abolished or severely altered by further shortening of either sequence. Remarkably, this minimal transcript contains, within its 18S rRNA part, long sequences complementary to both U20 and U13 small nucleolar RNAs (snoRNAs). The cis-acting elements essential for the reaction were studied further by site-directed mutagenesis. The U20 snoRNA complementary region in 18S rRNA was not required for faithful processing at the 18S rRNA/ITS 1 junction. Also, processing at this site was not appreciably altered by random substitution of proximal ITS 1 sequences (including the 5' terminal nucleotide) or of the terminal nucleotide of mature 18S rRNA. Substitutions in the four-nucleotide loop of the 18S rRNA 3'-terminal stem-loop, including the two adenosine residues substrates of dimethylation, did not alter appreciably the formation of the 18S rRNA 3' end, showing that the (methyl)2A1850.(methyl)2A1851 doublet was not required for processing at this site. Two highly conserved 18S rRNA elements acted as major cis-acting signals for processing at the 3' end, the CAUU sequence immediately preceding the 3'-terminal nucleotide and the 3' strand of the 3'-terminal 18S rRNA helix, complementary to U13 snoRNA. Compensatory mutations, restoring the potential for helix formation, but not U13 snoRNA complementarity, did not restitute the cleavage at the 3' end of 18S rRNA. This suggests that U13 snoRNA may be a trans-acting factor in the nucleolytic cleavage at the 3' end of 18S rRNA.

The in vivo hypersensitivity of eukaryotic rRNA gene transcription to actinomycin D has long been known, but this effect could not be reproduced in model systems and its molecular mechanisms remain uncertain. We studied the action of actinomycin D using mouse rRNA minigenes (with RNA polymerase I promoter and terminator signals), carrying truncated mouse or human rDNA inserts, which are faithfully transcribed upon transient transfection into mouse cells. Low concentrations (0.01-0.08 micrograms/ml) of actinomycin D caused within 1-2 h a 2-7-fold stimulation of the transcription of rRNA minigenes which is inversely related to the size of the rDNA transcript. With transcripts longer than 3 kb the effect was reversed and at 4 kb a practically complete inhibition of the formation of full-length transcripts was observed, accompanied, however, by an enhanced accumulation of unfinished rDNA transcripts. The dependence of actinomycin D action on transcript length was also observed with lacZ gene segments of different size inserted into the mouse rRNA minigenes. The transcription initiation of endogenous rRNA genes was also stimulated by the low doses of actinomycin D as indicated by the enhanced synthesis of unfinished rDNA transcripts (spanning mainly the 5' external transcribed spacer), whereas the synthesis of full-length transcripts was abolished. Removal of actinomycin D from the medium caused within 8-24 h a dramatic increase of the transcription from all rRNA minigenes tested. This stimulation was also inversely related to the size of the transcripts and varied from twofold to fivefold for the 3-4-kb transcripts to about 50-80-fold for the basic minigene transcript (395 nucleotides). The amount of endogenous aborted rDNA transcripts was also markedly increased, but the synthesis of full-length transcripts was not restored even 24 h after removal of the drug. The present results reproduce in a model cellular system the in vivo hypersensitivity of rRNA gene transcription to actinomycin D and reveal that the major factor involved is the size of the rRNA gene transcript. This effect requires only the basic rRNA gene promoter and terminator signals and does not depend on the G + C content of the RNA polymerase I transcripts. We suggest that at low concentrations, the intercalation of actinomycin D changes the conformation of DNA in the promoter region in a manner that stimulates the transcription of both endogenous and transfected rRNA genes.(ABSTRACT TRUNCATED AT 400 WORDS)

The processing of pre-rRNA in eukaryotic cells involves a complex pattern of nucleolytic reactions taking place in preribosomes with the participation of several nonribosomal proteins and small nuclear RNAs. The mechanism of these reactions remains largely unknown, mainly because of the absence of faithful in vitro assays for most processing steps. We have developed a pre-rRNA processing system using the transient expression of ribosomal minigenes transfected into cultured mouse cells. Truncated mouse or human rRNA genes are faithfully transcribed under the control of mouse promoter and terminator signals. The fate of these transcripts is analyzed by the use of reporter sequences flanking the rRNA gene inserts. Both mouse and human transcripts, containing the 3' end of 18S rRNA-encoding DNA (rDNA), internal transcribed spacer (ITS) 1, 5.8S rDNA, ITS 2, and the 5' end of 28S rDNA, are processed predominantly to molecules coterminal with the natural mature rRNAs plus minor products corresponding to cleavages within ITS 1 and ITS 2. To delineate cis-acting signals in pre-rRNA processing, we studied series of more truncated human-mouse minigenes. A faithful processing at the 18S rRNA/ITS 1 junction can be observed with transcripts containing only the 60 3'-terminal nucleotides of 18S rRNA and the 533 proximal nucleotides of ITS 1. However, further truncation of 18S rRNA (to 8 nucleotides) or of ITS 1 (to 48 nucleotides) abolishes the cleavage of the transcript. Processing at the ITS 2/28S rRNA junction is observed with truncated transcripts lacking the 5.8S rRNA plus a major part of ITS 2 and containing only 502 nucleotides of 28S rRNA. However, further truncation of the 28S rRNA segment to 217 nucleotides abolishes processing. Minigene transcripts containing most internal sequences of either ITS 1 or ITS 2, but devoid of ITS/mature rRNA junctions, are not processed, suggesting that the cleavages in vivo within either ITS segment are dependent on the presence in cis of mature rRNA sequences. These results show that the major cis signals for pre-rRNA processing at the 18S rRNA/ITS 1 or the ITS2/28S rRNA junction involve solely a limited critical length of the respective mature rRNA and adjacent spacer sequences.

rRNA processing pathways in humans have been reinvestigated through systematic Northern-blot hybridizations of HeLa cell nuclear RNA with a collection of digoxigenin-labeled rDNA probes from different regions of the human rDNA transcriptional unit. In addition to the known 45S, 41S, 32S and 21S pre-rRNA, two major pre-rRNA fractions were identified; a '30S' (about 5800 nucleotides) precursor to 18S rRNA containing an external transcribed spacer at the 5' end (ETS) and internal transcribed spacer (ITS) 1 sequences and a '12S' (about 950 nucleotides) precursor to 5.8S rRNA containing ITS 2 sequences. These pre-rRNA species do not react with probes located near the 3'-terminal segments of ITS 1 or ITS 2, thus suggesting that processive endonuclease cuts occur within ITS spacer sequences. The simultaneous occurrence of at least two alternative 45S pre-rRNA processing pathways is deduced, which correspond to a different temporal order of endonuclease attack at the sites located near the 5' end of 18S rRNA and within ITS 1. In-vivo labeling experiments with [14C]uridine revealed that inhibition of protein synthesis with cycloheximide abolishes the endonuclease cut at the 5' end of 18S rRNA and the formation of 41S pre-rRNA, while the cut within ITS 1 and the processing to 32S and '30S' pre-rRNA remains relatively unaltered.

A gene encoding human basic fibroblast growth factor has been chemically synthesized, cloned and expressed in Escherichia coli as a biologically active protein. The 465 bp gene was assembled by enzymatic ligation of 6 pairs of oligonucleotides and cloned in the expression vector pLCII downstream from the strong PL promoter. This promoter directed the synthesis of a fusion protein between a 31 amino acids fragment of the lambda phage cII protein and bFGF. A four amino acid recognition sequence for the site-specific protease fXa was introduced in the plasmid construct and this allowed cleavage of the fusion protein at the boundary between cII and bFGF. bFGF was purified close to homogeneity using a Heparin-Sepharose column and Mono S cation exchange chromatography. The use of the pLCII expression system resulted in the accumulation of 20 to 25 mg of purified bFGF per l of bacterial culture. The recombinant bFGF was mitogenic for mouse 3T3 fibroblasts and the dose-response curve was similar to the one for native bFGF.

In regenerating rat liver both the transcriptional activity of the intergenic spacer rDNA promoter and the steady-state abundance of spacer transcripts are increased about 2-fold, as compared to normal liver. These changes are parallel to the observed 2.5-fold increase in regenerating liver of rRNA gene promotor activity and gene promotor transcripts abundance. These results suggest that both gene and spacer rDNA promotors are subject to common regulatory mechanisms. Our results indicate also that the stability of spacer transcripts in regenerating liver is not significantly altered.

Mitotin is a 125 kDa/pI 6.5 nuclear protein specific for proliferating cells and markedly increased prior to and during mitosis. This study presents evidence for the expression of this protein during dimethylsulfoxide (DMSO) induced differentiation of human promyelocytic leukemia HL 60 cells. The expression had been followed at two levels: as antigen, using a specific antimitotin monoclonal antibody and as mRNA, using a specific cDNA probe. The results from the immunofluorescent study show a gradual disappearance of mitotin in differentiating HL 60 cells starting from the fourth day after DMSO induction. On the other hand, the changes in the expression of mitotin mRNA were much more dramatic. This mRNA is expressed at a high level during the first three days of differentiation but shows a striking decrease after the fourth day. This correlates with the rapid changes in the number of blast cells in the differentiating HL 60 cell population. Therefore, the expression of mitotin mRNA can serve as a marker for the changes accompanying the termination of cell proliferation in differentiating cells.

The preparation of mammalian cells for entry into mitosis is related to a cascade of G2 phase phosphorylations of several nuclear proteins driven by mitosis-specific protein kinases. Using a monoclonal antibody we have identified previously in mammalian cells a 125K/pI6.5 protein, associated with the nuclear matrix, and markedly increased in mitotic cells, which was named 'mitotin'. Here, we show by short-term [35S]methionine labeling of cell cycle synchronized cells that this protein is synthesized at comparable rates throughout interphase. However, upon cycloheximide block of protein synthesis mitotin labeled during S phase is rapidly degraded, while the degradation of mitotin labeled during late G2 phase is abolished, resulting in its net and marked increase. The accumulation of mitotin in premitotic and mitotic cells is related to its phosphorylation and the metabolic stability of its two phosphorylated forms. The metabolic stabilization and accumulation of a nuclear matrix protein upon phosphorylation suggests the operation of a novel mechanism among the complex events preparing the cell for mitosis.

alpha-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of alpha-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of alpha-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10-20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by alpha-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.

An adenosine-utilizing mutant of Saccharomyces cerevisiae (SY 15 ado) is isolated after remutagenesis of an osmotic-sensitive strain, auxotrophic for adenine, with ethyl methanesulfonate. It is shown that the SY15ado mutant can be used to achieve experimental conditions under which cell growth and RNA Synthesis are directly dependent on exogenous adenosine. After starvation for adenosine, toyocamycin is incorporated into pre-rRNA chains of SY15ado cells replacing adenosine residues. The extent of this replacement depends on the concentration of added toyocamycin. Lower doses slow down processing of pre-rRNA into mature rRNA with an accumulation of 27 S and 20 S pre-rRNA. At higher concentrations toyocamycin blocks the last steps of pre-rRNA processing i.e. the conversions 27 S pre-rRNA leads to 25 S rRNA and 20 S pre-rRNA leads to 18 S rRNA. It appears that the main site of toyocamycin action is at the last steps of ribosome formation, while transcription and the early stages of pre-RNA processing are less affected.

rRNA from detergent-purified nuclei was fractionated quantitatively, by two independent methods, into nucleolar and nucleoplasmic RNA fractions. The two RNA fractions were analysed by urea/agar-gel electrophoresis and the amount of pre-rRNA (precursor of rRNA) and rRNA components was determined. The rRNA constitutes 35% of total nuclear RNA, of which two-thirds are in nucleolar RNA and one-third in nucleoplasmic RNA. The identified pre-rRNA components (45 S, 41 S, 39 S, 36 S, 32 S and 21 S) are confined to the nucleolus and constitute about 70% of its rRNA. The remaining 30% are represented by 28 S and 18 S rRNA, in a molar ratio of 1.4. The bulk of rRNA in nucleoplasmic RNA is represented by 28 S and 18 S rRNA in a molar ratio close to 1.0. Part of the mature rRNA species in nucleoplasmic RNA originate from ribosomes attached to the outer nuclear membrane, which resist detergent treatment. The absolute amount of nuclear pre-rRNA and rRNA components was evaluated. The amount of 32 S and 21 S pre-rRNA (2.9 x 10(4) and 2.5 x 10(4) molecules per nucleus respectively) is 2-3-fold higher than that of 45 S, 41 S and 36 S pre-rRNA.

Nucleolar '80-S' and '40-S' preribosomes (containing 45-S and 21-S pre-rRNA, respectively), as well as cytoplasmic ribosomes, were isolated from Friend erythroleukemia cells. The presence of structural ribosomal proteins in the isolated particles was studied by using antisera against individual rat liver small ribosomal subunit proteins. The analysis is based on the established crossreactivity between rat and mouse ribosomes [F. Noll and H. Bielka (1970) Mol. Gen. Genet. 106, 106-113]. The identification of the proteins was achieved by two independent immunological techniques: the passive haemagglutination test and the enzyme immunoassay of electrophoretically fractionated proteins, blotted on nitrocellulose. All 17 proteins tested are present in cytoplasmic ribosomes. A large number of proteins (S3a, S6, S7, S8, S11, S14, S18, S20, S23/24 and S25) are present in the '80-S' preribosome. Only two proteins (S3 and S21) are added during the formation of the '40-S' preribosome in the nucleolus. Four proteins (S2, S19, S26 and S29) are added at later, possibly extranucleolar, stages of ribosome formation. The results obtained provide evidence for the sequential addition of proteins during the formation of the small ribosomal subunit in Friend erythroleukemia cells.

We investigated the organization of the rat rDNA non-transcribed spacer (NTS) by determining the sequence of large NTS segments located upstream (2501 bp) and downstream (4025 bp) from the rRNA transcription unit. We identified four B2-like and two ID mobile elements. They may be grouped in three pairs with the members of each pair located in the upstream and downstream NTS. The ID sequences are identical to the consensus sequence, while the pairs of B2-like elements show 85% and 50/65% homology to the consensus B2 sequence. The proximal part of the downstream NTS contains a region of widely diverged SalI tandem repeats. A considerable part of the analyzed upstream and downstream NTS sequences is constituted by different types of simple sequences and long poly(purine) X poly(pyrimidine) tracts. These data show that the rat rDNA NTS regions flanking the rRNA transcription unit are characterized by a combination of short interspersed (B2-superfamily) and various simple sequences.

The 5'-terminal of Saccharomyces cerevisiae 18 S and 25 S rRNA are precisely mapped within the sequence of the rDNA repeating unit. The 3'-terminal of 25 S rRNA and 37 S pre-rRNA are located within a 548 bp segment of the rDNA repeating unit by the use of a DNA polymerase I extension technique. The analysis of the rDNA sequences at the structural gene boundaries reveals the presence of oligonucleotide repeats which may be involved in transcription or processing control mechanisms. The sequence of rDNA in the transcription termination region is determined and possible mechanisms shaping the 3'-end of 25 S rRNA are discussed.

The action of low (5 mg/kg body wt;) and high (20 mg/kg body wt.) doses of cycloheximide, both causing a rapid and almost complete inhibition of protein synthesis in rat liver is investigated. Short-term (15 min) [14C]orotate incorporation into nucleolar rRNA in vivo is inhibited only by the high dose acting for periods longer than 1 h. The effect may be correlated with a strongly reduced labelling of the cellular pool of free uridine nucleotides. These results indicate that in vivo transcription of rRNA genes may not be under stringent control. The activity of template-bound RNA polymerase A in nuclei isolated from animals treated with both doses of cycloheximide is reduced within 1 h to about 50% of controls reaching nearly plateau levels at longer times of action of the drug. The differential effect of cycloheximide inhibition of protein synthesis on in vivo and in vitro rRNA synthesis suggests the existence of elongation control protein(s) characterized by a rapid turnover and a loose association with the nucleus.

The complete nucleotide sequence of the 25 S rRNA gene from one rDNA repeating unit of Saccharomyces cerevisiae has been determined. The corresponding 25 S rRNA molecule contains 3392 nucleotides and has an estimated relative molecular mass (Mr, Na-salt) or 1.17 x 10(6). Striking sequence homology is observed with known 5'- and 3'-end terminal segments of L-rRNA from other eukaryotes. Possible models of interaction with 5.8 S rRNA are discussed.

The pre-rRNA and rRNA components of rat and mouse liver nucleolar RNA were analysed. It was shown that upon denaturation, part of the 32 S pre-rRNA is converted into 28 S rRNA and 12 S RNA. The 12 S RNA from mouse (Mr, 0.36 X 10(6)) is larger than the one from rat (Mr, 0.32 X 10(6). The 12 S RNA chain is intact and resists denaturation treatment. The non-covalent binding of this RNA with nucleolar 28 S rRNA is stronger than that of 5.8 S rRNA with 28 S rRNA. Hybridization with a rat internal-transcribed spacer rDNA fragment identifies 12 S RNA as corresponding to the 5'-end non-conserved segment of 32 S pre-rRNA, including 5.8 S rRNA. The significance of the formation of a 12 S precursor to 5.8 S rRNA in the biogenesis of ribosomes in mammalian cells is discussed.

The endonuclease cleavage of 41 S pre-rRNA to yield 32 S and 21 S pre-rRNA constitutes a major early step in the processing of pre-rRNA in rat liver. The 5'-terminus of 32 S pre-rRNA and the 3'-terminus of 21 S pre-rRNA were precisely located within the rDNA sequence by S1 nuclease protection mapping and use of appropriate rDNA restriction fragments. The 5'-terminus of 12 S pre-rRNA, an initial product of 32 S pre-rRNA processing, was also mapped within the rDNA sequence. The 5'-termini of 32 S and 12 S pre-rRNA coincide and map within a 14-residue T-tract (non-coding strand) at 161-163 bp upstream from the 5'-end of the 5.8 S rRNA gene. The 3'-terminus of 21 S pre-rRNA maps within the same T-tract. These results show that the endonuclease cleavage occurs within a U-tract in the internal transcribed spacer 1 sequence of 41 S pre-rRNA. The homogeneity of the 5'- or 3'-termini of 32 S, 12 S and 21 S pre-rRNA indicates also that the terminal processing of these molecules, if any, is markedly slower. The coincidence in the location of 32 S and 12 S pre-rRNA 5'-termini shows further that the endonuclease cleavage of 32 S pre-rRNA precedes the removal of its 5'-terminal segment to yield 5.8 S rRNA. The absence in the whole pre-rRNA internal transcribed spacer of sequences complementary to the target U-tract suggests that the endonuclease cleavage, generating 32 S and 21 S pre-rRNA, occurs in a single-stranded loop of U-residues.

D-Galactosamine (250 mg/kg body weight) causes 90--95% inhibition of [14C]orotate or inorganic [32P]phosphate incorporation in vivo into rat liver nuclear RNA within 30 min. The transcription of both nucleolar and nucleoplasmic genes is inhibited to the same extent. Under these conditions, prelabbeled 45-S pre-rRNA is processed quantitatively to nuclear 28-S and 18-S rRNA. The nucleocytoplasmic transport of both 28-S and 18-S rRNA remains unaltered for about 60 min after blockage of transcription. At this stage the nucleo-cytoplasmic transport of 18-S rRNA is almost completed. It is concluded that formation and nucleo-cytoplasmic transport of ribosomes is independent of concurrent transcription of rRNA or nucleoplasmic genes. At later stages, the nucleocytoplasmic transport of 28-S rRNA is delayed and its partial degaradation in the nucleus may take place. This effect is correlated with a decreased (up to 40% of controls) labelling of nuclear proteins. However, the labelling of total cellular or microsomal proteins remains unchanged up to 3 h after D-galactosamine administration. It is suggested that the last nuclear steps of ribosome formation are dependent on the continuous supply of rapidly-labelled nuclear proteins.

In previous studies of proliferating mammalian cells a p125/6.5 nuclear matrix antigen displaying a marked increase in mitotic cells has been identified. This antigen was investigated by immunocytochemistry of cryosections of testes at different stages of postnatal development: newborn, 20 days after birth and sexually mature rats. In Sertoli cells, the distribution of the p125/6.5 antigen parallels [3H]thymidine incorporation: present in newborn and absent in sexually mature testes. The p125/6.5 antigen is present also in some prespermatogonia of the newborn rat testis, which do not incorporate [3H]thymidine. At later stages of development, the p125/6.5 antigen is present also in first meiotic prophase spermatocytes displaying an extrachromosomal nucleoplasmic distribution, while absent in spermatids and spermatozoa. These results show that the p125/6.5 antigen increases not only during mitosis, but also during meiosis. They suggest further that this antigen is characteristic of both proliferating cells and cells (prespermatogonia) committed to proliferation.

An anti-nucleolar monoclonal antibody reacting with human B-lymphoblastoid cells but not with normal peripheral blood lymphocytes has been isolated. The antibody recognized in Namalwa cells an antigen with molecular mass 41 kDa and pI 5.6, different from all previously described nucleolar antigens. Inhibition of rRNA transcription with Actinomycin D caused redistribution of the 41/5.6 antigen, but even at long term drug action it remains associated with the nucleolar remnants.

The possible sites of 5.8S:25S rRNA interaction in Saccharomyces cerevisiae are investigated by blot-hybridization of in vivo 32P-labelled 5.8S rRNA with restriction fragments from the 25S rRNA gene. Strong hybridization signals are obtained with fragments from the 5'-end (nucleotides 1 to 494) and the 3'-end (3066 to 3391) of the gene. The fragments from the remaining part of the gene are negative. A computer analysis of the known Saccharomyces cerevisiae 5.8S (Rubin 1973) and 25S (Georgiev et al. 1981) rRNA sequences show a markedly higher complementarity between 5.8S rRNA and the 5'- and 3'-terminal segments of 25S rRNA corresponding to the hybridization positive rDNA fragments. In accordance with the experimental and sequence analysis data, two alternative end-to-end base pairing models of possible 5.8S:25S rRNA binding are proposed. Both models imply that 5.8S rRNA connects the 5'- and 3'-terminal segments of 25S rRNA, but differ in the extent of preservation of the secondary structure typical of free 5.8S rRNA. It is suggested that the requirement for 5'- and 3'-end binding of 23S rRNA in Escherichia coli is conserved in evolution and that in eukaryotes 5.8S rRNA plays the role of joining together the two ends of L-rRNA molecules.

The method for electron microscopy of the Ag-staining of NOR-specific proteins was adapted to tissue sections from solid organs. The distribution of the Ag-staining proteins in rat liver nucleoli after complete block of transcription caused by D-galactosamine was investigated. In control animals, the Ag-staining proteins are associated with the fibrillar components of nucleoli. After block of transcription, positive Ag-staining is observed in the condensed fibrillar components of segregated nucleoli and later in the derived dense nucleolar fibrillar remnants. The granular components and the spherical bodies in segregated nucleoli are negative. It is concluded that in interphase nucleoli the Ag-staining NOR proteins are associated with the fibrillar components and with the derived nucleolar fibrillar remnants. The positive Ag-staining does not reflect the actual transcription of rRNA genes since it is present in both transcribed and non-transcribed r-chromatin.

The initial endonuclease cleavage site in 32 S pre-rRNA (precursor to rRNA) is located within the rate rDNA sequence by S1-nuclease protection mapping of purified nucleolar 28 S rRNA and 12 S pre-rRNA. The heterogeneous 5'- and 3'-termini of these rRNA abut and map within two CTC motifs in tSi2 (internal transcribed spacer 2) located at 50-65 and 4-20 base-pairs upstream from the homogeneous 5'-end of the 28 S rRNA gene. These results show that multiple endonuclease cleavages occur at CUC sites in tSi2 to generate 28 S rRNA and 12 S pre-rRNA with heterogeneous 5'- and 3'-termini, respectively. These molecules have to be processed further to yield mature 28 S and 5.8 S rRNA. Thermal-denaturation studies revealed that the base-pairing association in the 12 S pre-rRNA:28 S rRNA complex is markedly stronger than that in the 5.8 S:28 S rRNA complex. The sequence of about one-quarter (1322 base-pairs) of the 5'-part of the rat 28 S rDNA was determined. A computer search reveals the possibility that the cleavage sites in the CUC motifs are single-stranded, flanked by strongly base-paired GC tracts, involving tSi2 and 28 S rRNA sequences. The subsequent nuclease cleavages, generating the termini of mature rRNA, seem to be directed by secondary-structure interactions between 5.8 S and 28 S rRNA segments in pre-rRNA. An analysis for base-pairing among evolutionarily conserved sequences in 32 S pre-rRNA suggests that the cleavages yielding mature 5.8 S and 28 S rRNA are directed by base-pairing between (i) the 3'-terminus of 5.8 S rRNA and the 5'-terminus of 28 S rRNA and (ii) the 5'-terminus of 5.8 S rRNA and internal sequences in domain I of 28 S rRNA. A general model for primary- and secondary-structure interactions in pre-rRNA processing is proposed, and its implications for ribosome biogenesis in eukaryotes are briefly discussed.

Cycloheximide given in vivo at low doses (2--5 mg/kg body weight) causes within 30 min a complete inhibition of protein synthesis in rat liver. The labelling of nuclear proteint is also strongly inhibited. Under these conditions, the amount of nucleolar 45-S pre-rRNA and its [14C]-orotate labelling remain unaffected for at least 4 h. These results show that initially the rates of synthesis and processing of 45-S pre-rRNA are not appreciably altered. On the other hand, drastic alterations in the 45-S pre-rRNA processing pathways occur at the early stages of cycloheximide action. Formation of 18-S rRNA is abolished and that of 28S rRNA is reduced to about half the level in control rats. This dichotomy in the production of the two ribosomal particles may be correlated with a block in the formation of 41-S and 21-S pre-rRNA. Generation of 36-S and 32-S pre-rRNA is still taking place, but the rate of their processing to nucleolar 28-S rRNA is decreased, thus causing the accumulation of these two pre-rRNA species. In parallel, processing of 45-S pre-rRNA to an aberrant 39-S rRNA species is markedly enhanced. The results obtained show that the channelling of nucleolar pre-rRNA along alternative processing pathways is under stringent control by the continuous supply of critical protein(s).

Ribonucleic acid (RNA) synthesis in the sorbitol-dependent, fragile yeast mutant VY1160 (Venkov et al., 1974) is rapidly inhibited by rifampin. The growth of the mutant cells and protein synthesis are more slowly affected by the antibiotic, apparently as secondary phenomena. Lower doses of rifampin (50 to 100 mug/ml) preferentially inhibit ribosomal RNA synthesis in comparison to that of messenger RNA and transfer RNA. Transcription and translation of messenger RNA continues in the presence of low doses of rifampin, as evidenced by the unimpaired induction of alpha-glucosidase. Partially purified RNA polymerase II from this mutant, in contrast to that from the parental strain, is strongly inhibited by low concentrations (1 mug/ml) of rifampin, whereas RNA polymerase I from the two strains is similar in behavior. The mutant may be useful for the study of regulatory mechanisms of transcription in eukaryotes.

A monoclonal antibody of the IgG2a subclass was isolated from the supernate of a hybridoma line obtained with splenocytes from a mouse immunized with a crude nucleolar fraction of human Namalwa cells. This antibody identifies a single nuclear polypeptide antigen characterized by: (a) presence in proliferating human cell lines and phytohemagglutinin-stimulated lymphocytes, but absence in resting lymphocytes; (b) appearance in stimulated lymphocytes in parallel with the onset of DNA synthesis; (c) a speckled distribution in the nucleoplasm; (d) tight association with nuclear matrix structures identified by both biochemical and in situ extraction and enzyme treatment procedures; (e) mol wt of 125 kDa and pI 6.5 as determined by immunoprecipitation or immunoblotting of nuclear or nuclear matrix proteins fractionated by gel electrophoresis. The above characteristics identify the p125/6.5 nuclear matrix protein recognized by the isolated monoclonal antibody as belonging to the class of proliferating cell nuclear antigens.

A method for the rapid isolation of active transcription complexes from animal cell nuclei is described. The method is based on the observation that, after lysis of nuclei with the detergents Sarkosyl and Triton X-100, transcription complexes are selectively bound to nitrocellulose. The nitrocellulose filters retain 80-90% o the RNA labelled briefly in vitro and about 10% of the nuclear DNA. The bulk of the retained DNA is in the size range of 20 kb. Transcription complexes involving both RNA polymerase I and II are retained by nitrocellulose. The nitrocellulose-bound transcription complexes preserve almost all of their RNA polymerase activity. The size distribution of the RNA product shows that bound transcription complexes retain also most of their growing RNA chains. The possibility to use selective retention by nitrocellulose in the analysis of transcriptionally active genes is discussed.

The maturation of pre-rRNA (precursor to rRNA)in liver nuclei is studied by agar/ureagel electrophoresis, kinetics of labelling in vivo with [14C] orotate and electron-microscopic observation of secondary structure of RNA molecules. (1) Processing starts from primary pre-rRNA molecules with average mol. wt. 4.6X10(6)(45S) containing the segments of both 28S and 18S rRNA. These molecules form a heterogeneous peak on electrophoresis. The 28S rRNA segment is homogeneous in its secondary structure. However, the large transcribed spacer segment (presumably at the 5'-end) is heterogeneous in size and secondary structure. A minor early labelled RNA component with mol.wt. about 5.8X10(6) is reproducibly found, but its role as a pre-rRNA species remains to be determined. (2) The following intermediate pre-rRNA species are identified: 3.25X10(6) mol.wt.(41S), a precursor common to both mature rRNA species ; 2.60X10(6)(36S) and 2.15X10(6)(32S) precursors to 28S rRNA; 1.05X10(6) (21S) precursor to 18S rRNA. The pre-rRNA molecules in rat liver are identical in size and secondary structure with those observed in other mammalian cells. These results suggest that the endonuclease-cleavage sites along the pre-rRNA chain are identical in all mammalian cells. (3) Labelling kinetics and the simultaneous existence of both 36S and 21S pre-rRNA reveal that processing of primary pre-rRNA in adult rat liver occurs simultaneously by at least two major pathways: (i) 45S leads to 41S leads to 32S+21S leads to 28S+18S rRNA and (ii) 45S leads to 41S leads to 36S+18S leads to 32S leads to 28S rRNA. The two pathways differ by the temporal sequence of endonuclease attack along the 41 S pre-rRNA chain. A minor fraction (mol.wt.2.9X10(6), 39S) is identified as most likely originating by a direct split of 28S rRNA from 45S pre-rRNA. These results show that in liver considerable flexibility exists in the order of cleavage of pre-rRNA molecules during processing.

The sequence of one cloned rat rDNA segment is determined, encompassing the 3'-terminal part of 18 S rDNA (231 bp), ITS 1 (1072 bp), 5.8 S rDNA (156 bp), ITS 2 (771 bp) and the 5'-terminal part of 28 S rDNA (210 bp). Comparison with a distinct clone of the same rDNA segment reveals the identity of the rRNA gene sequences and considerable heterogeneity in both ITS 1 and ITS 2. The heterogeneities include mainly single base-pair changes (23 deletions or insertions and 14 substitutions) distributed all along the ITS sequences.

We studied the behaviour in interphase and mitotic human cells of a 125 kDa (pI 6.5) antigen, associated with the nuclear matrix and detected in proliferating cells. Indirect immunofluorescence with a specific monoclonal antibody reveals that during interphase in WISH and Namalwa cells, as well as phytohaemagglutinin-stimulated lymphocytes, the antigen displays a speckled distribution in the nucleoplasm of all cells. At early prophase the fluorescence intensity of the coalesced speckles increases markedly. During metaphase and anaphase the antigen gives maximal fluorescence distributed diffusely in the nucleoplasm, while chromosomes remain negative. At anaphase and cytokinesis the antigen is still cytoplasmic, but fluorescence intensity decreases. Two-dimensional gel electrophoresis and immunoblotting reveal that the p125/6.5 antigen displays a net increase in isolated mitotic cells as compared to interphase cells. These results suggest that the p125/6.5 protein participates in late G2 phase and G2/M transition events preparing the cell for mitosis.

The 5'-terminal coding sequence for the 37 S precursor to rRNA of Saccharomyces cerevisiae is identified by reverse transcriptase extension and protection mapping with nuclease S1. The sequence of a 419 bp rDNA fragment containing the transcription initiation site and its adjacent region is determined.

The reaction product of the ribosomal poly(A) polymerase [ATP(UTP):RNA nucleotidyltransferase] is analyzed. Two systems are used in vitro: (a) isolated polyribosomes with endogenous enzyme and RNA primer and (b) purified enzyme with total polyribosomal RNA as primer. In the polyribosome system about 50% of the [3H]AMP label is in poly(A)-containing mRNA. This RNA displays a heterogeneous size ditribution in the range of 8--30 S with a maximum at about 14 S. Upon denaturation the maximum is shifted towards the 10-S zone. The poly(A) polymerase catalyzes the addition of 12--18 adenylate residues to pre-existing mRNA poly(A) sequences of 40--160 residues. The [3H]AMP incorporated into poly(A)-lacking RNA is mainly in a fraction with an electrophoretic mobility corresponding to 4-S RNA. In the purified enzyme system, specificity towards poly(A)-containing mRNA is lost to a considerable extent. Only 10% of the [3H]AMP label is retained by oligo(dT)-cellulose. The bulk of the product is in 18-S rRNA and heterogeneous small molecular weight RNA. We conclude that the ribosome-associated poly(A) polymerase is most likely the enzyme responsible for the cytoplasmic polyadenylation of poly(A)-containing mRNA in vivo.

A series of prototrophic fragile strains of different ploidy (2n, 3n and 4n) has been genetically constructed on the basis of haploid srb1 containing segregants of the fragile Saccharomyces cerevisiae mutant VY 1160. The strains have been characterized by several criteria. In regard to generation time, biomass yield, and nucleic acids content of the cells, the tetraploid srb1 homozygous hybrid is indistinguishable from an industrial strain of S. cerevisiae. However, it is characterized by a higher protein content. Unlike any other laboratory or industrial strains, the original mutant and these hybrids possess an ability for lysis upon suspension in hypotonic solutions. The dependence of the percentage of lysed cells on the growth phase and concentration of osmotic stabilizer in the medium has been investigated. The quantity of proteins in the soluble and insoluble fractions obtained after lysis of these strains by osmotic shock has been determined. These hybrids can be considered as a potential industrial source of proteins for nutritional purposes.

The incorporation of [14C]orotate and [14C]uridine into UMP residues of hnRNA (heterogeneous nuclear RNA) and pre-rRNA (precursors to rRNA) of Eharlich ascites-tumour cells was compared: orotate was incorporated at a markedly higher rate into hnRNA. On the other hand, the rate of incorporation of uridine into pre-rRTNA was even somewhat higher than into hnRNA. The ratio of specific radioactivities of CMP to UMP residues in pre-rRNA and hnRNA was studied. At all times of labelling this ratio was similar for both RNA species independently of the precursor used. On addition of excess unlabelled uridine, the CMP/UMP labelling ratio in both pre-rRNA and hnRNA rose. However, this increase was much more pronounced with hnRNA. It is concluded that nuclear pyrimidine nucleotide pool for RNA synthesis is compartmentalized. The synthesis of hnRNa is supplied preferentially by the large and the small compartment, respectively. A detailed model for the cellular compartmentation of uridine nucleotide precursors to RNA is proposed.U

The primary structure of rat (Rattus norvegicus) 28 S rRNA is determined inferred from the sequence of cloned rDNA fragments. The rat 28 S rRNA contains 4802 nucleotides and has an estimated relative molecular mass (Mr, Na-salt) of 1.66 X 10(6). Several regions of high sequence homology with S. cerevisiae 25 S rRNA are present. These regions can be folded in characteristic base-paired structures homologous to those proposed for Saccharomyces and E. coli. The excess of about 1400 nucleotides in the rat 28 S rRNA (as compared to Saccharomyces 25 S rRNA) is accounted for mainly by the presence of eight distinct G+C-rich segments of different length inserted within the regions of high sequence homology. The G+C content of the four insertions, containing more than 200 nucleotides, is in the range of 78 to 85 percent. All G+C-rich segments appear to form strongly base-paired structures. The two largest G+C-rich segments (about 760 and 560 nucleotides, respectively) are located near the 5'-end and in the middle of the 28 S rRNA molecule. These two segments can be folded into long base-paired structures, corresponding to the ones observed previously by electron microscopy of partly denatured 28 S rRNA molecules.

The synthesis and processing of RNA by isolated HeLa cell nuclei was studied at low ionic strength in the presence of alpha-amanitin. The RNA polymerase reaction, with endogenous template and enzyme, rapidly reaches a plateau dependent on the amount of nuclei. Evidence is presented that incorporation of [(3)H]UMP proceeds only in growing RNA chains, whereas initiation of new RNA chains is arrested. The product formed contains all the main components of the 45S pre-rRNA (precursor of rRNA) maturation pathway (45S, 32S and 20S pre-rRNA; 28S and 18S rRNA). Most of the labelled material is in the mature rRNA components and their immediate precursors, even at very short times of incubation (2min). Small, but definite, 5S and 4S RNA peaks are also observed. At shorter incubation times a substantial amount of [(3)H]UMP is incorporated into RNA molecules in the 24S and 10-16S zones. This RNA material is considered to represent the non-conserved segments of 45S pre-rRNA in the process of nucleolytic degradation. A model for the tracer study of the topology of 45S pre-rRNA, on arrest of rRNA initiation, is discussed. The experimental evidence obtained supports the following structure of 45S pre-rRNA: 5'-end-28S rRNA unit-18S rRNA unit-nonconserved segment-3'-end.

A monoclonal antibody (MAb) identifies the nuclear antigen p125/6.5 associated with the nuclear matrix and present in proliferating human cells. By direct and indirect immunofluorescence, the presence and distribution of the antigen p125/6.5 in cryostat sections from primary and metastatic solid human tumors has been investigated. The antigen is present in nuclei of malignant and benign tumor cells, but is not detected in adjacent normal tissues. The antigen displays a speckled nucleoplasmic distribution, while nucleoli are negative. The intensity of the immunofluorescence reaction is markedly higher in the nuclei of some individual or grouped tumor cells.

The kinetics of in vivo labeling of cellular free UMP and nucleolar, nucleoplasmic, and cytoplasmic rRNA with [14C]orotate in rat brain and liver were investigated. Evaluation of the experimental data shows: (a) The rate of nucleolar precursors of ribosomal RNA (pre-rRNA) synthesis and the deduced rate of ribosome formation in brain is about fivefold lower than in liver and corresponds to 220-260 ribosomes/min/nucleus. (b) The lower rate of in vivo pre-rRNA synthesis is correlated with a lower activity of RNA polymerase I in isolated brain nuclei. (c) The half-lives of nucleolar rRNA in brain and liver are 210 and 60 min, respectively, thus showing a slower rate of processing of pre-rRNA in brain nucleoli. (d) The nucleo-cytoplasmic transport of ribosomes in brain is also markedly slower than in liver and reflects the lower rates of synthesis and processing of pre-rRNA. (e) Cytoplasmic ribosomes in brain and liver turn over with half-lives of about 6 and 4 days, respectively. It is concluded that the markedly lower rate of ribosome biogenesis in brain is specified mainly at the level of transcription of rRNA genes.